Molecular Imaging Using Labeled Donor Tissues Reveals Patterns of Engraftment, Rejection, and Survival in Transplantation

Cao, Yu; Bachmann, Michael; Beilhack, Andreas; Yang, Yang; Tanaka, Masashi; Swijnenburg, Rutger-Jan; Reeves, Robert E.; Taylor-Edwards, C; Schulz, Stephan; Doyle, Timothy C.; Fathman, C. Garrison; Robbins, Robert C.; Herzenberg, Leonore A.; Negrin, Robert S.; Contag, Christopher H.

doi:10.1097/01.tp.0000164347.50559.a3

Cited by 97 publications

(99 citation statements)

References 20 publications

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“…35 This mouse expresses hMGFP under the control of the chick b-actin (CAG) promoter (Supplementary Figures 1a, b see also Gonzalez-Gonzalez 35 ), which also contains CMV enhancer elements. A similar suprabasal pattern of reporter expression has been observed following intradermal plasmid injections 38,[51][52][53] as well as in transgenic animals, 54,55 where transcription is driven by CMV or CAG promoters. To further investigate the involvement of genetic elements from CMV in the observed expression pattern, additional hMGFP constructs were prepared that use other promoters (Ubc, EF1a and eIF4A1) that lack CMV elements.…”

Section: Nuclei Are Visualized With Dapi (Blue)mentioning

confidence: 53%

Increased interstitial pressure improves nucleic acid delivery to skin enabling a comparative analysis of constitutive promoters

González-González

Spitler

et al. 2010

Gene Ther

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Nucleic acid-based therapies hold great promise for treatment of skin disorders if delivery challenges can be overcome. To investigate one mechanism of nucleic acid delivery to keratinocytes, a fixed mass of expression plasmid was intradermally injected into mouse footpads in different volumes, and reporter expression was monitored by intravital imaging or skin sectioning. Reporter gene expression increased with higher delivery volumes, suggesting that pressure drives nucleic acid uptake into cells after intradermal injections similar to previously published studies for muscle and liver. For spatiotemporal analysis of reporter gene expression, a dual-axis confocal (DAC) fluorescence microscope was used for intravital imaging following intradermal injections. Individual keratinocytes expressing hMGFP were readily visualized in vivo and initially appeared to preferentially express in the stratum granulosum and subsequently migrate to the stratum corneum over time. Fluorescence microscopy of frozen skin sections confirmed the patterns observed by intravital imaging. Intravital imaging with the DAC microscope is a noninvasive method for probing spatiotemporal control of gene expression and should facilitate development and testing of new nucleic acid delivery technologies.

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Section: Nuclei Are Visualized With Dapi (Blue)mentioning

confidence: 53%

Increased interstitial pressure improves nucleic acid delivery to skin enabling a comparative analysis of constitutive promoters

González-González

Spitler

et al. 2010

Gene Ther

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“…The difference between the amount of luciferin injected and of photons detected is in part a reflection of the amount of injected luciferin that has access to the intracellular enzyme (34), the uniformity of luciferase expression in different cell types (33,42), the number of luciferin molecules turned over by luciferase, the quantum efficiency of the enzyme, and the absorption and scattering of the signal by mammalian tissues. The photon flux thus represents a minimum but reproducibly quantifiable measure of uptake and release.…”

Section: Figmentioning

confidence: 99%

Real-time analysis of uptake and bioactivatable cleavage of luciferin-transporter conjugates in transgenic reporter mice

Wender

Goun²,

Jones³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Many therapeutic leads fail to advance clinically because of bioavailability, selectivity, and formulation problems. Molecular transporters can be used to address these problems. Molecular transporter conjugates of otherwise poorly soluble or poorly bioavailable drugs or probes exhibit excellent solubility in water and biological fluids and at the same time an enhanced ability to enter tissues and cells and with modification to do so selectively. For many conjugates, however, it is necessary to release the drug/probe cargo from the transporter after uptake to achieve activity. Here, we describe an imaging method that provides quantification of transporter conjugate uptake and cargo release in real-time in animal models. This method uses transgenic (luciferase) reporter mice and whole-body imaging, allowing noninvasive quantification of transporter conjugate uptake and probe (luciferin) release in real time. This process effectively emulates drug-conjugate delivery, drug release, and drug turnover by an intracellular target, providing a facile method to evaluate comparative uptake of new transporters and efficacy and selectivity of linker release as required for fundamental studies and therapeutic applications.drug delivery ͉ imaging ͉ transgenic animals M olecular transporters § are agents which, when covalently linked to or complexed with a cargo, enable or enhance its entry into cells or tissues. Many types of transporters have been reported in recent years including peptides (1-5), peptoids (6), polyamines (7,8), oligocarbamates (9), dendrimers (10, 11), polysaccharides (12), steroids (13), cationic lipids (14, 15), guanidinoglycosides (16), and even nanotubes (17). These transporters operate through a variety of mechanisms and some through multiple mechanisms depending on cell type, cargo, and other variables (5,(18)(19)(20)(21)(22). Among the classes of transporters, oligoguanidine based transporters are particularly promising, providing excellent water solubility and at the same time the remarkable ability to rapidly cross the nonpolar membrane of a cell. These transporters have been used for the delivery of small molecules, peptides, proteins, nucleic acids, liposomes, and imaging agents (23-29) and have been modified to provide selective delivery of drugs into target cells and tissue (30,31). Significantly, an octaarginine transporter has been shown to facilitate uptake into tissue (32), including human skin (23). A conjugate of octaarginine and Cyclosporin A enabling uptake of the latter selectively into the skin and thereby effectively eliminating its systemic toxicity has been advanced into Phase II clinical trials (23).There are two overarching and interrelated challenges confronting the further advancement of this field: the development of methods to quantify the uptake of new or existing transporters in real-time in animal models and the identification and evaluation of linkers that would allow for controllable release (if required) of a free drug/probe from the transporter conjugate only after cel...

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“…Mice transgenic for firefly luciferase were initially developed in the laboratory of Dr. Robert Negrin 5 . Because of their constitutive luciferase expression, cells from these donor mice can be tracked in vivo using a non-invasive imaging technique 5 . Another advantage of this model is that individual mice can be monitored over time.…”

Section: Discussionmentioning

confidence: 99%

Induction of Graft-versus-host Disease and <em>In Vivo</em> T Cell Monitoring Using an MHC-matched Murine Model

Anthony

Hadley

2012

JoVE

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Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo.Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity).Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant.Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

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Molecular Imaging Using Labeled Donor Tissues Reveals Patterns of Engraftment, Rejection, and Survival in Transplantation

Cited by 97 publications

References 20 publications

Increased interstitial pressure improves nucleic acid delivery to skin enabling a comparative analysis of constitutive promoters

Increased interstitial pressure improves nucleic acid delivery to skin enabling a comparative analysis of constitutive promoters

Real-time analysis of uptake and bioactivatable cleavage of luciferin-transporter conjugates in transgenic reporter mice

Induction of Graft-versus-host Disease and <em>In Vivo</em> T Cell Monitoring Using an MHC-matched Murine Model

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